Fluorescent probes are valuable reagents for the analysis and separation of molecules and cells and for the detection and quantification of other materials. A very small number of fluorescent molecules can be detected under optimal circumstances. Barak and Webb visualized fewer than 50 fluorescent lipid analogs associated with the LDL reception of cells using a SIT camera, J. CELL BIOL. 90:595-604 (1981). Flow cytometry can be used to detect fewer than 10,000 fluorescein molecules associated with particles or certain cells (Muirhead, Horan and Poste, BIO/TECHNOLOGY 3:337-356 (1985)). Some specific examples of the application of fluorescent probes are (1) identification and separation of subpopulations of cells in a mixture of cells by the techniques of fluorescence flow cytometry, fluorescence-activated cell sorting and fluorescence microscopy; (2) determination of the concentration of a substance that binds to a second species (e.g., antigen-antibody reactions) in the technique of fluorescence immunoassay; and (3) localization of substances in gels and other insoluble supports by the techniques of fluorescence staining. These techniques are described by Herzenberg et al., “CELLULAR IMMUNOLOGY” 3rd ed., Chapter 22; Blackwell Scientific Publications (1978); and by Goldman, “FLUORESCENCE ANTIBODY METHODS” Academic Press, New York, (1968); and by Taylor et al., APPLICATIONS OF FLUORESCENCE IN THE BIOMEDICAL SCIENCES, Alan Liss Inc. (1986).
When employing fluorescent enzyme substrates for the above purposes, there are many constraints on the choice of a fluorescent enzyme substrate. One constraint is the absorption and emission characteristics of the fluorophore generated from a fluorescent enzyme substrate, since many ligands, receptors, and materials in the sample under test, e.g. blood, urine, cerebrospinal fluid, will fluoresce and interfere with an accurate determination of the fluorescence of the fluorescent label. This phenomenon is called autofluorescence or background fluorescence. Another consideration is the ability to keep the fluorescent enzyme substrates and their enzymatic products inside of cells through the conjugation of cellular components with the reactive fluorescent substrates and/or their enzymatic products. A third consideration is the quantum efficiency of the products generated from the enzyme substrates which should be high for sensitive detection. A fourth consideration is the light absorbing capability, or extinction coefficient, of the fluorescent products derived from the reaction of the enzyme substrates, which should also be as large as possible.
The applicability and value of the methods indicated above are closely tied to the availability of suitable fluorescent enzyme substrates. In particular, there is a need for fluorescent substances that can be excited by commercial viable laser sources such as the violet laser (405 nm), argon laser (488 nm) and He—Ne laser (633 nm). There are many fluorescent enzyme substrates developed for the argon laser (488 nm excitation) and He—Ne laser (633 nm excitation). For example, fluorescein-based enzyme substrates, which are well excited by 488 nm argon laser, are useful emitters in the green region. CFSE, a reactive fluorescein-based esterase substrate, is widely used for monitoring cell proliferations with argon laser excitation, e.g., Asquith et al., Proc. Biol. Sci., 2006, 273, 1165; Cao et al., Cytometry A, 2009, 75, 975; Lyons, J. Immunol. Methods, 2000, 243, 147; Witkowski, in “Current Protocols in Cytometry” Chapter 9, Unit 925. However, there are few reactive fluorescent enzyme substrates available for the 405 nm violet laser. Brightly fluorescent enzyme substrates permit detection or location of the attached materials with great sensitivity. Certain coumarin enzyme substrates have demonstrated utilities for a variety of biological detection applications, e.g., U.S. Pat. No. 6,566,508 to Bentsen et al. (2003); U.S. Pat. No. 6,207,404 to Miller et al. (2001); U.S. Pat. No. 5,830,912 to Gee et al. and U.S. Pat. No. 4,956,480 to Robinson (1990).